| Starch is the main storage form of carbohydrates in plants,which can be widely used as food and industrial raw materials for people and animals.In plants,starch exists in the form of semi crystalline granules(starch granules,SG),including storage starch and temporary starch.They play an important role in plant growth,development and response to abiotic stress.Storage starch is accumulated in the seed,storage root and other storage organs of plants.Temporary starch is present in chloroplasts and is usually synthesized during the day and degraded at night.At present,there are many researches on the shape and size of maize endosperm SG and the proteins related to starch biosynthesis and degradation,but due to the difficulty of separating pollen and starch granule in leaves,there are few related researches,especially the comparative analysis of the characteristics of these three SG in maize is not clear.In this article,the two-phase method of ethanol/NaH2PO4(ATS)was established for the separation and purification of starch grains from maize seeds,pollen and leaves.The purity of SG in seeds,pollen and leaves was detected by scanning electron microscopy,projection electron microscopy and immunoblotting.The SG binding protein was identified and analyzed by 2-DE,tandem mass spectrometry and bioinformatics.The morphological characteristics,physical and chemical properties and protein composition were compared.The results showed that there were significant differences in morphological characteristics,physicochemical properties and protein composition of SG in seeds,pollen and leaves.The endosperm SG is spherical or polygonal,with an average size of 15.4 μm;the pollen SG is rod-shaped,with an average size of 2.29 μm;the leaf SG is pie shaped,with an average size of 1.90 μm.The contents of amylose in endosperm,pollen and leaf SG were 27.6%,18.9%and 8.1%,respectively.The gelatinization temperature of pollen SG(74.0℃)was higher than that of endosperm(69.5℃)and leaf SG(70.0℃).SDS-PAGE analysis showed that the band pattern of SG binding protein in leaves was different from that in seeds and pollens.2-DE analysis showed that there were significant differences in SG binding protein between endosperm and pollen,of which 38 and 27 protein spots were identified.These proteins were mainly various starch synthetases.The endosperm SG contained more binding starch synthetase and starch branching enzyme,while the pollen SG contained more starch synthetase and no starch branching enzyme.In conclusion,a rapid and effective method of separating starch granules from plants was established in this article,and the morphological characteristics,physical and chemical properties and protein composition of three kinds of starch in maize were compared.The results of this article can provide a new clue for further understanding the characteristics and synthesis process of starch granules,and play an important role in improving the physical and chemical properties and functional research of starch. |
